9/8/17. Fetal Alcohol Spectrum Disorder & Direct Alcohol Biomarkers. How I Became Involved. How I Became Involved. Douglas Lewis, DSc, USDTL

Transcription

1 Fetal Alchl Spectrum Disrder & Direct Alchl Bimarkers Duglas Lewis, DSc, USDTL Gateway Technical Cllege Racine, WI Octber 6, 2017 Hw I Became Invlved 2 Hw I Became Invlved CMH/NU Feinberg Schl f Medicine

2 9/8/17 Befre I Became Invlved 4 FASD & Direct Alchl Bimarkers Hw I Became Invlved in This Jurney CMH/NU Feinberg Schl f Medicine 1983 Dr Ira Chasnff/ Perinatal Center f Chemical Dependence Ccaine use is uniquely damaging t a fetus, far mre damaging than ther narctics - Ira Chasnff, MD

3 9/8/17 S What Was The Prblem? 7 S What Was The Prblem? Ccaine was nt the culprit, binge alchl drinking was! 8 Then What Was The Slutin? A lng-term marker fr fetal binge alchl expsure was desperately needed. 9 3

4 Direct Alchl Bimarkers Three nn-xidative EtOH metablite(s) emerged Ethyl Glucurnide (EtG) Fatty Acid Ethyl Esters(FAEE) Phsphatidylethanl (PEth) 10 FASD & Direct Alchl Bimarkers Ethyl Glucurnide 11 Ethyl Glucurnide Ethyl Glucurnide first t be characterized R.T. Williams identified and ran TLC 1953 Rabbits Rec'd pure standard frm Dr Artz, Crn Prducts, Arg, IL Derivatized as triacetyl b-ethyl-d-glucurnide methyl ester Williams had EtG ready fr GC analysis, waited fr instrumentatin t arrive 1995 EtG GC analysis described 12 4

5 Ethyl Glucurnide GC/MS EI f triacetyl b-ethyl-d-glucurnide methyl ester Cncentrated frm urine f alchl user 13 Ethyl Glucurnide Chrmatgraphic Cnditins: HP 5971 GC with HP 5988 MS Detectr Clumn: Chrmpack CP Sil-5 (12 m x 0.25 mm) Injectr Temp: 250 deg Clumn Prgram Initial Temp: 140 deg x 1 min Ramp 20 deg/min Final Temp: 320 deg x 1 min In selected in mnitring mde LOD 100 ng/ml 14 Ethyl Glucurnide Methd invlved simple cncentratin f 1 ml urine Needed dual derivatizatin t frm methyl, acetyl derivative. A time cnsuming prcess, but a step frward 15 5

6 FASD & Direct Alchl Bimarkers Fatty Acid Ethyl Esters - Ethyl Stearate 16 Fatty Acid Ethyl Esters Fatty acid ethyl esters described 1991 Lapasta and Lange USDTL began develpment in mecnium 1992 Extractin prved difficult Ply-unsaturated FAEE s lw recvery (<1%) NIAAA funded SBIR Phase I Grant 1996 Breakthrugh 1998 Lapsata serum methd Extractin with an aminprpyl SPE High ply-unsaturates recveries First frensic test fr fetal alchl expsure 17 Fatty Acid Ethyl Esters 18 6

7 Fatty Acid Ethyl Esters Chrmatgraphic Cnditins: Agilent 5973 GC/MS System Ps CI Methane Clumn: SGE BPX-70 (30 m x 0.25 mm) Injectr Temp: 250 deg Clumn Prgram Initial Temp: 100 deg x 1 min Ramp 20 deg/min Final Temp: 260 deg x 1 min In selected in mnitring mde LOD 50 ng/gram 19 Fatty Acid Ethyl Esters FAEE analysis extended t hair specimens Eurpean labs especially favred Over time, majr issues arse FAEE s cntained in csmetic hair prducts EtOH n hair made FAEE s in hair EtOH wipes n mecnium made FAEE s in mecnium 20 FASD & Direct Alchl Bimarkers Phsphatidylethanl (PEth) Phsphatidylethanl (PEth) discvered in 1983 Abnrmal phsphlipid that frms when: EtOH + Phsphatidylchline + Phsphlipase D all are present All human smatic cells cntain Phsphatidylchline + Phsphlipase D 21 7

8 Phsphatidylethanl (PEth) PEth can frm in red bld cells as a cmpnent f the cellular membrane. PEth is a direct alchl bimarker, meaning that ethanl is incrprated int the final prduct. 22 Phsphatidylethanl (PEth) Early PEth analyses used HPLC-ELSD HPLC-ELSD is relatively insensitive Assay culd nly detect HEAVY drinking Liquid/liquid extractin als a drawback Breakthrughs in LC/MS/MS changed PEth 23 Phsphatidylethanl (PEth) In the early 2000 s LC/MS/MS sensitivity increased many fld. PEth became available at cutffs 10 x lwer than HPLC-ELSD Whle bld PEth nw culd detect a single binge drinking event fr ver a week. 24 8

9 Phsphatidylethanl (PEth) With the arrival f ultra high sensitivity LC/MS/MS systems came: Sample size reduced t dried bld spts n filter paper NO venipuncture! Newbrn Specimens culd be analyzed 25 Phsphatidylethanl (PEth) DBS vs. Whle Bld Easier Cllectin Shipped as nn-bihazard Less rm needed fr strage Extractin quicker, safer and cleaner 26 Micr Liquid Chrmatgraphy (Micr-LC) Eksigent Micr-LC/ AB SCIEX Triple Quad 5500 Eksigent Hal 3.0 minute methd 50 µl /min methd Apprximately 0 sq ft LC space 324 vial capacity ALS utlets needed fr LC LOQ 8ng/mL Analyst Sftware/Eksigent driver Operatr friendly 27 9

10 Micr LC - 50 µl/min Micr-LC LC Prgram Time A% B% A= 50% 2mM Ammnium Acetate/25% Acetnitrile/25% Isprpanl B= 60% Acetnitrile/40% Isprpanl 28 Micr LC-MSMS LOQ 8ng/mL 733 > > > > FASD & Direct Alchl Bimarkers The reductin f specimen size t DBS als led t: Newbrn Heel Stick specimens Newbrn umbilical crd bld sampling The prblems f FAEE analysis in mecnium are gne The sensitivity as a evidentiary test is superir The nursing staff are far mre cmpliant We still can t believe hw well it wrks! A definitive study is currently funded by NIAAA an is nging 30 10

11 Fingernail Testing fr Alchl With the arrival f ultra high sensitivity LC/MS/MS systems als came: EtG in fingernails Why fingernails? It is anther keratin matrix, like hair Unlike hair, it grws in tw dimensins, length and thickness 31 Anatmy f Fingernail Nail Plate Nail Matrix Nail Bed 32 FASD & Direct Alchl Bimarkers We btained 606 paired hair and fingernail specimens frm cllege age subjects EtG in fingernails averaged 2.5 x the hair cncentratins EtG in fingernails shwed n gender bias, hair did. May be due t female use f csmetic prducts n hair. N difference in cmpliance

12 Cllectin f Specimens NIAAA 1 R44 AA (Phase II) UW- Milwaukee- CABHR Hair Specimen Nail Specimen 34 FASD & Direct Alchl Bimarkers Summary We have cme a very lng way since 1953 We nw have validated frensic tests that prvide: 3 Day windw Urine EtG (and EtS) 3 Week windw PEth 3 Mnth windw Fingernail/Hair EtG 35 FASD & Direct Alchl Bimarkers Summary All f these advancements are the results f: Instrument manufacturer s cnstant imprvements in LC/MS/MS Academic researchers explring new cmpunds and pathways Prviders wh take chances and implement these new ideas 36 12

13 FASD & Direct Alchl Bimarkers Where d the Direct Alchl Bimarkers fit int identifying FASD? 37 FASD Diagnstic Algrithm. H. Eugene Hyme et al. Pediatrics di: /peds by American Academy f Pediatrics 38 FASD & Direct Alchl Bimarkers Mms self reprt alchl use in pregnancy rarely. When Mms reprt alchl use, they almst always underreprt usage. PEth des nt cnfabulate. PEth has yet t have a dcumented False Psitive 100% Specificity. The PEth result fr newbrns is a vice fr a viceless 39 13

14 FASD & Direct Alchl Bimarkers Why use PEth? Extremely Sensitive Already DBS specimens are cllected PEth in DBS are highly stable and easily stred 40 FASD & Direct Alchl Bimarkers PEth analysis psitivity has varied by regins Midwest state snapsht fund 4% Psitive Sutheast State snapsht fund 20+% Psitive Mntevide, Uruguay hspital randm study fund 85+% Psitive Drinking cultures vary, s d newbrn results 41 FASD & Direct Alchl Bimarkers NIAAA funded U01 study in Mntevide, Uruguay and Sa Paul, Brazil will allw lngitudinal study f relatinship f PEth and FASD First study f its kind t prvide this kind f data Dr. Philip May s Active Surveillance Methd will be used t assess FASD

15 FASD & Direct Alchl Bimarkers We ve cme a lng way frm the Ccaine Babies f the 1980 s It tk ver 30 years t find a bimarker and the analytical system t reliably use it t identify fetal alchl expsure. This next step f studying the relatinship f expsure t disease is the mst exciting f all. 43 Thank Yu All Rights Reserved. Cpyright 2017 United States Drug Testing Labratries, Inc. 15